{"paper_id":"45e4c590-3d34-42a3-87d5-1118ddd38ff4","body_text":"Tuina alleviates the muscle atrophy of sciatic nerve injury rats through regulating PI3K/Akt signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tuina alleviates the muscle atrophy of sciatic nerve injury rats through regulating PI3K/Akt signaling 英奇 张, 羽羽 张, 嘉悦 刘, 佳伟 周日, 岳 徐, Narentuya shi, 洪正 张, 嘉旺 燕, 金平 陈, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4839192/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Dec, 2024 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted 7 You are reading this latest preprint version Abstract Background Tuina has been shown to be an effective treatment for the decrease of skeletal muscle atrophy after Peripheral nerve injury (PNI). However, its mechanism is unclear. This study aimed to explore the underlying mechanisms of tuina on rats with sciatic nerve injury (SNI). Methods A rat model of SNI was established. After a total of 20 times tuina intervention, the curative effects were evaluated by behavioral assessment, nerve function index and muscle atrophy index (MAI). The pathological changes were observed by transmission electron microscopy and immunofluorescence. The levels of IGF-1 and FoxO were detected by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to detect the expression of proteins in the PI3K/AKT signaling pathway. Result The behavioral assessment, nerve function index and MAI proved that tuina significantly improved muscle atrophy after SNI compared with that in SNI model group. Transmission electron microscopy showed that tuina improved ultramicrostructure and immunofluorescence of CD31 showed that tuina improved microcirculation. Further, we observed that tuina differentially regulated levels of IGF-1 and FoxO and the protein expression of p-PI3K, p-AKT and VEGF in anterior tibial muscle and soleus muscle. Conclusion Tuina could effectively inhibit skeletal muscle atrophy via the microcirculation pathway in the rat model of SNI, by regulating the expression levels of IGF-1, FoxO. The underlying mechanism of action might be via the PI3K/Akt signaling pathway. Peripheral nerve injury Tuina Muscle atrophy Microcirculation PI3K/AKT pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Peripheral nerve injury (PNI), a neurological condition that seriously threatens both human and animal patients[ 1 ], a big percentage of which is associated with poor functional outcomes, insufficient nerve recovery, and the loss of motor function. These are followed by partial recovery, muscle atrophy, and profound weakness[ 2 ]. The change of microvascular after nerve injury is an important reason for muscle atrophy. Injury-induced loss of local blood vessels contribute to inflammation and ischemia and thus to the overall damage to the nerve and even muscle that innervate[ 3 ]. The reversal of microvascular dysfunction may provide new approaches for the treatment and prevention of PNI diseases. Skeletal muscle atrophy is a debilitating consequence of denervation, of which is one of the contributing factors towards incomplete functional recovery[ 4 ]. A combination of factors, including increased proteolysis, decreased protein synthesis, and impaired regenerative capacity, contributes to skeletal muscle atrophy [ 5 ]. The activation of phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway prevents muscle atrophy through inhibiting the activity of FoxO transcription factors and augmenting protein synthesis. PI3K/Akt signaling can also dominantly inhibit the effects of myostatin causing an increase in skeletal muscle size[ 6 ]. Moreover, PI3K, as well as its downstream target Akt, has been implicated in a number of cellular responses linked to angiogenesis, including endothelial cell migration and survival. Overexpression of constitutively active PI3K or Akt promotes angiogenesis in vivo and increases VEGF expression[ 7 ]. Tuina, as a type of non-surgical interventions, is an alternative medical therapy which is safe and has virtually no side effects. Accumulating literature have reported its application in multiple diseases improving disability and pain in patients[ 8 – 10 ]. Additionally, due to its anti-inflammatory and blood circulation-promoting effects, Tuina manipulations are widely used to myopathy recovery[ 11 ]. Studies have confirmed that Tuina has a good effect on the treatment of PNI through autophagy, synaptic plasticity, axon regeneration, and remyelination, and ultimately achieve the purpose of restoring sensory and motor function[ 12 – 14 ]. In conclusion, whether the mechanical effect of tuina on PNI inhibit skeletal muscle atrophy via the microcirculation pathway remains unknown. Additionally, whether this process is regulated by the PI3K/AKT pathway deserves further exploration. To address these questions, we established a rat model of SNI and observed the effects of tuina on muscle function, muscle atrophy, microcirculation and PI3K/AKT pathway. Materials and methods Animals The animal protocol was approved by the Animal Care and Use Committee of the Beijing University of Chinese Medicine (No. BUCM-2023032303-1119). All animal experiments were designed by the principles of the 3Rs (Replacement, Reduction and Refinement) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague–Dawley (SD) rats (6-week-old, 190–210 g) were purchased from Beijing SPF Biotechnology Co., Ltd. (Beijing, China), and dwelled in a pathogen-free environment with 4 animals per cage. The rats are fed in a 12-h light-dark cycle environment, temperature (25 ± 0.5) ℃, humidity 40% − 50%, and have free access to diet and drinking water. SNI model 36 rats were randomly divided into four groups (n = 9): Blank Control (CON) group, Sham Model (SHA) group, SNI Model (SNI) group and Tuina (TUI) group. Pathological modeling started after one week of acclimatization. The rat model of SNI was established as previously described[ 13 , 14 ]. After anesthetized, an incision of about 1 cm long was made in the skin along the direction of the sciatic nerve exposing the lower edge of the piriformis. For the rats in SNI and TUI groups, the special hemostatic pliers were used to clamp 5 mm at the distal end of the sciatic nerve nodule for 5 s with full force (6 N) resulting in a length of about 2 mm injury point. For the rats in SHA group, the sciatic nerve was only found, sterilized, sutured, and then sterilized. Tuina intervention The TUI group received “Three-Manipulation and Three-Acupoint” treatment, the procedure was performed as follows: The Tuina Manipulation Simulator (Self-developed machine, China invention patent number ZL202320511277.5) was set to stimulate with a force of 4 N, 60 times per minute (Fig. 1 B). The stimulus rod was placed on BL 37, GB 34, and BL 57 of the surgical side, then finger pressing, plucking, and kneading manipulation were stimulated, respectively. Each acupoint and manipulation were operated for 1 min consecutively for 9 min in total. There were ten times treatments following 1 day rest and ten times treatments were repeated, so that a total of 20 times treatments were completed. The grip restraint intervention was performed in the SHA and SNI groups. In order to reduce animal stress response, petting and stroking animals for 9 min before the formal intervention every day. Behavioral assessment An electrically inclined plate tester was used to detect the changes in the muscle strength of the rats’ hind limbs. Rats in each group were taken for behavioral testing and the test was measured at baseline before the surgery, on the day of 10-time and 20-time after the intervention. The heads of the rats were placed on the board toward the end and gradually increase the angle of the board after the rats calmed down. When the rats cannot stay in this position for 5 s, the critical angle of the protractor was recorded, and the average level of three measurements was taken. Nerve function index Nerve function index were collected using the DigiGait™ Imaging System and then analyzed by the DigiGait™ 15.0 analysis software (Mouse Specifics, Inc.; Quincy, MA, USA). The rats were habituated to the apparatus 1 week before the experiment, with the treadmill belt gradually accelerated to 10 cm/s. Muscle atrophy index (MAI) After the rats were sacrificed, the anterior tibial muscle and soleus muscle were removed, and their wet weights were measured using an electronic balance. MAI is defined by muscle weight divided by body weight. The empty stomach weights of the rat pre-dissection and their anterior tibial muscle and soleus muscle (post-dissection) were measured. Transmission electron microscopy observation The anterior tibial muscle, soleus muscle and sciatic nerve were removed, fixed in pre-cooled 2.5% (w/v) glutaraldehyde for 3 h, 0.1M PB washed. Then were postfixed in a 1% (w/v) osmic acid solution for 1 h, 0.1M PB washed for 1h, dehydrated (through a grade series of ethanol solutions) and embedded in Epon 812 epoxy resin. The segments were cut into 70nm-thick ultrathin slices. Then the sections were stained with saturated aqueous uranyl acetate (2%) and citrate and were observed and analyzed by TEM. Immunofluorescence The anterior tibial muscle, soleus muscle and sciatic nerve were fixed in 4% paraformaldehyde at 4 ◦C, dehydrated using a sucrose gradient, embedded in optimal cutting temperature compound, and cut into 4um sections. Paraffin sections were dewaxed, dehydrated, subjected to antigen retrieval, cleared of spontaneous fluorescence, and blocked with serum. Primary antibodies for CD 31(1:500, abcam, USA) were given for incubation at 4 ℃ overnight. Sections were rinsed with phosphate buffered saline and incubated with appropriate secondary antibodies at 37℃ for 30min, then rinsed with phosphate buffered saline. The sections were observed under a fluorescence microscope, and Image J was used to analyze the Pearson coefficient of immunofluorescence colocalization and the fluorescence intensity of each protein. Enzyme-linked immunosorbent assay (ELISA) 50 milligrams of the gastrocnemius muscle and tibialis anterior muscle were weighed, and PBS was added at a weight (mg)/volume (µL) ratio of 1:10 for homogenization. protein concentrations of the samples were determined with the ELISA kit. The test procedures were as follows: standard dilution, sample addition, washing, color development, reaction termination, then the absorbance of each well was measured, and finally calculated the concentration of FoxO, and IGF-1 by drawing a standard curve. Western blotting The gastrocnemius muscle, tibialis anterior muscle and sciatic nerve were lysed in RIPA lysis buffer. The BCA protein assay kit was used to measure the protein concentration. Briefly, 20 µg of total protein was separated by SDS-PAGE and transferred to PVDF membranes at 400 mA for 30 min. Subsequently, the membranes were blocked for 30min at room temperature with Blocking solution. The membranes were incubated overnight at 4°C with the following primary antibodies: mouse anti-GAPDH (1:10000, Ym3029, Immunoway), rabbit anti-p-PI3K (1:1000, AF3241, Affbiotech), rabbit anti-p-Akt (1:1000, bs-0867R, Bioss), and rabbit anti-VEGF (1:1000, bs-1665R, Bioss). After washing three times with TBST, the secondary HRP-conjugated antibodies were as follows: goat anti-rabbit IgG H&L (1:10000, bs-0295G, Bioss), goat anti-mouse IgG H&L (1:10000, bs-0296G, Bioss). After washing three times with TBST, an ECL kit was used to detect the immunoactivity. Statistical analysis Data analysis was performed with SPSS Statistics software Version 26.0 (IBM). Results were presented as mean ± SD. Student’s t-test was used to compare differences between two groups. One-way ANOVA was used for comparisons between groups, and the LSD multiple comparison test was used for multiple comparisons. P < 0.05 was treated as statistically significant. Results Tuina improved behavioral performance There was no redness or swelling point of injury for rats in each group, and the health status was good after the modeling operation. After the SNI model was established, the fine motor function was greatly affected, indicating that the model was successfully prepared. For behavioral assessment, the angle of the inclined plate was used to evaluate the muscle strength and motor function. And the results showed that the angle of the inclined plate of the rats in the CON and SHA group before intervention, after 10-time and 20-time interventions were not statistically difference. Compared with the SHA group, the inclined plate angle of the rats in the SNI group and the TUI group decreased significantly before intervention but there was no significant difference between the SNI group and the TUI group. Compared with those in the SNI group after the 10-time and 20-time interventions, the inclined plate angle of the rats in the TUI group increased significantly, but there were still significant differences compared with those in the SHA group. Those results showed that tuina intervention can effectively improve the motor function in the lower limbs after nerve injury (Fig. 1 C). Tuina promoted the repair of nerve injury The nerve function index was used to evaluate the recovery of injury-nerve. The results of rats in each group showed that, compared with the baseline, the Sciatic function index (SFI), Tibial function index (TFI), and Peroneal function index (PFI) of rats in the SNI group and the TUI group decreased significantly before intervention; compared with the SNI group after the 10-time and 20-time interventions, the SFI, TFI, PFI of rats in the TUI group increased significantly. Those results showed that tuina intervention can effectively promote the recovery of fine movements in the hind limbs of rats and facilitate the recovery of motor function of nerve injury (Fig. 1 D). A: animal experimental flow; B: Tuina intervention; C: The angle of the inclined plate changes; D: The nerve function index; D1: SFI; D2: TFI; D3: PFI. CON: control; SHA: sham; SNI: sciatic nerve injury; TUI: Tuina. Results are presented as mean ± standard deviation. ** P < 0.001 vs SHA; ## P < 0.001 vs SNI. Tuina alleviated muscle atrophy Muscle atrophy index (MAI) was used to evaluate the muscle atrophy. The MAI of the anterior tibial muscle and soleus muscle had the similar changes among four groups. Results of the rats in each group showed that, there were no statistically differences in the CON and SHA group. Compared with the SHA group, the MAI of the rats in the SNI group and the TUI group decreased significantly. Compared with the SNI group, the TUI group increased significantly, but there were still significant differences compared with those in the SHA group. Those results showed that tuina intervention can effectively relieve muscle atrophy caused by nerve damage (Fig. 2 C). Transmission electron microscopy observation was used to evaluate the ultrastructural changes of muscles. Results of the anterior tibial muscle and soleus muscle showed that, in the CON and SHA groups: the myosin filaments were arranged neatly and the transverse lines were obvious. A band was formed by interlacing and overlapping heterotropic myosin filaments and actin filaments. Only the actin filaments pass through the region, showing an isotropic I band; there were regular Z lines between I band; obvious sarcomeres between the Z line junctions. The M line is the site of marked thickening of myosin. A large number of mitochondria were present among the muscle fibers, mitochondrial morphology normal and regular. In the SNI group: the myofilaments were arranged disorderly and the horizontal lines disappeared. Typical structures of skeletal muscle such as A band, I band, Z line, M line disappeared. The space between muscle bundles was significantly enlarged. A large number of vacuolated mitochondria were found between muscle bundles and muscle fibers. In the TUI group: the myofilaments were arranged neatly and horizontal lines appeared. Clearly visible lines to Z line and M line, A band and I band could identify. The internal structure of mitochondria between myofilaments and fascicles was not clear, and occasionally vacuolated mitochondria were observed. Those results showed that tuina intervention can effectively relieve ultrastructural changes of muscle atrophy caused by nerve damage (Fig. 2AB) A: ultramicrostructure of anterior tibial muscle; A1, A2, A3, A4: at low magnification; Scale bars: 1 µm; A5, A6, A7, A8: mitochondria at high magnification; Scale bars: 200 nm; A1, A5: CON group; A2, A6: SHA group; A3, A7: SNI group; A4, A8: TUI group; B: ultramicrostructure of the soleus muscle; B1, B2, B3, B4: at low magnification; Scale bars: 1 µm; B5, B6, B7, B8: mitochondria at high magnification; Scale bars: 1 µm; B1, B5: CON group; B2, B6: SHA group; B3, B7: SNI group; B4, B8: TUI group; C: MAI. C1: MAI of anterior tibial muscle; C1: MAI of soleus muscle. MAI: muscle atrophy index. Results are presented as mean ± standard deviation. ** P < 0.001 vs SHA; ## P < 0.001 vs SNI. Tuina enhanced microcirculation Immunofluorescence was used to evaluate the microcirculation of muscles and nerve. Figure 3 shows the results of immunofluorescence staining for CD 31 from each group. The nuclei were stained blue with DAPI, and CD 31 was stained red and marked microvascular endothelial cells. The expression of CD 31 in sciatic nerve, anterior tibial muscle and soleus muscle had the similar changes among four groups. Compared with CON and SHA group, the expression of CD 31 in the SNI group was significantly reduced. Compared with that in the SHA group, the fluorescence intensity of CD 31 in the SNI group was significantly decreased. A CD 31 distribution was observed in the TUI group, and the fluorescence intensity was significantly higher in the TUI group than in the SNI group. Those results showed that tuina intervention can effectively increase microvascular density after nerve injury in rats (Fig. 3ABC). A: Immunofluorescence staining of sciatic nerve; Scale bars: 100 µm; A1, A2, A3, A4: DAPI staining; A5, A6, A7, A8: CD 31 staining; A9, A10, A11, A12: merge of DAPI and CD 31 staining; A1, A5, A9: CON group; A2, A6, A10: SHA group; A3, A7, A11: SNI group; A4, A8, A12: TUI group; B: Immunofluorescence staining of anterior tibial muscle; Scale bars: 100 µm; B1, B2, B3, B4: DAPI staining; B5, B6, B7, B8: CD 31 staining; B9, B10, B11, B12: merge of DAPI and CD 31 staining; B1, B5, B9: CON group; B2, B6, B10: SHA group; B3, B7, B11: SNI group; B4, B8, B12: TUI group; C: Immunofluorescence staining of soleus muscle; Scale bars: 100 µm; C1, C2, C3, C4: DAPI staining; C5, C6, C7, C8: CD 31 staining; C9, C10, C11, C12: merge of DAPI and CD 31 staining; C1, C5, C9: CON group; C2, C6, C10: SHA group; C3, C7, C11: SNI group; C4, C8, C12: TUI group. Results are presented as mean ± standard deviation. * P < 0.05 vs SHA; ** P < 0.001 vs SHA; # P < 0.05 vs SNI. Tuina regulated muscle atrophy associated with IGF-1 and FoxO As shown in Fig. 4 , the anterior tibial muscle and soleus muscle expression levels of IGF-1were significantly lower in the SNI group and TUI group compared with the SHA group. Conversely, the TUI group displayed an increase in the expression levels of IGF-1compared with the SNI group. The expression levels of FoXO were significantly higher in the SNI group compared with the SHA group. The TUI group displayed a decrease in the expression levels of FoXO compared with the SNI group. These outcomes suggested that tuina could alleviate the muscle atrophy after SNI associated with IGF-1 and FoxO (Fig. 4AB). Tuina regulated factors associated with the PI3K/AKT pathway Western blotting was used to evaluate the changes of anterior tibial muscle and soleus muscle in the PI3K/AKT signaling pathway. The results revealed that the protein expression levels of p-PI3K and p-AKT significantly decreased in the SNI group. However, tuina intervention enhanced the expression. The protein expression levels of VEGF significantly increased in the TUI group, comparing with SNI. This suggested that tuins had a certain effect on the PI3K/AKT signaling pathway in the anterior tibial muscle and soleus muscle of rats with SNI (Fig. 5ABCD) Discussion Tuina, a non-pharmacological therapy with little or no side effects, has been extensively used in clinical practice for the relief and treatment of diseases. Under the guidance of traditional chinese medicine and western medicine anatomy and pathology, tuina acts on the body surface by various manipulations to regulate the physiological and pathological state, so as to treat diseases. Studies have shown that the benefits of tuina therapy include increased blood flow, reduced pain and perceived fatigue, attenuated inflammatory signals of muscle injury, improved muscle strength and self-perception of muscle injury, reduced muscle tension[ 15 – 17 ]. Tuina has advantages of not causing dependence, easier use, faster effect onset, and increased popularity among worldwide people including athletes. Related studies involve case reports, meta-analyses, controlled clinical trials, and animal experiments showed its positive effect on all kinds of injury. In this study, we found that tuina intervention could improve SNI rats hindlimb motor function by promoting nerve repair and inhibiting muscle atrophy. SNI model was used for simulating clinical PNI, which allows the evaluation of neuropathic changes and nerve regeneration. The “Three-Manipulation and Three-Acupoint” is a combination of manipulations and acupoints that we have studied and proven to be effective. Yinmen (BL 37) is located in the body surface projection area of the sciatic nerve trunk, at the location of biceps femoris muscle; Chengshan (BL 57) is located in the body surface projection area of the tibial nerve, at the location of gastrocnemius muscle and Yanglingquan (GB 34) is located in the body surface projection area of the common peroneal nerve, at the location of tibialis anterior muscle. We found that after 20-time intervention, the function and structure of muscles and nerve in the intervention area were significantly improved, and demonstrated significant effects of the “Three-Manipulation and Three-Acupoint”. Sciatic damage was estimated to contribute to 90% inhibition of muscle mass. Moreover, soleus muscles retained only 8% contractibility in 14-week denervated rats, the tibial anterior experienced 3–5% contraction in long-term denervation [ 18 ]. Evidence had shown that tuina could effectively accelerate the recovery of muscle fibers, improve the structure and morphology of gastrocnemius muscle, and delay the atrophy after sciatic nerve transection[ 19 , 20 ]. In this study, we found that SNI model led to the mass of anterior tibial muscle and soleus muscle decrease and ultrastructure change, and after tuina intervention, the mass of muscles increased significantly and ultrastructure restored obviously. Mitochondria play important roles in maintaining cellular homeostasis and skeletal muscle health, mitochondrial dysfunction can lead to skeletal muscle atrophy. During muscle atrophy, mitochondrial degradation influences the reduction of mitochondrial quality and quantity, which is controlled by mitochondrial autophagy as well as mitochondrial fusion and fission kinetics[ 21 ]. Therefore, in the strategy of treating muscle atrophy diseases, mitochondrial is the key entry point. Crane et al. showed that tuina therapy appears to be clinically beneficial by promoting mitochondrial biogenesis[ 22 ]. In this study, the results of electron microscope showed that the structure and morphology of mitochondrial is better in SNI rats treated with tuina than SNI model rats. The PI3K/Akt pathway is one of the most important pathways that regulates muscle atrophy. Muscle atrophy usually leads to the loss of muscle mass and function and is characterized by a reduction in muscle fiber size and mass, and an imbalance between protein synthesis and degradation in the muscle. Protein synthesis in skeletal muscle is mainly regulated by the PI3K/Akt signaling axis. This pathway plays a critical role in myotube hypertrophy, and activation of Akt in rat muscle prevents denervation-induced atrophy[ 23 ]. Additionally, an increase in the levels of the PI3K/Akt signaling pathway is critical for autophagy suppression, the formation of autophagosomes is stimulated by decreased levels of PI3K[ 24 ]. Skeletal muscle loses its contractile function after denervation, resulting in reduced blood perfusion and, thus, leading to nutritional and metabolic abnormalities in the target muscle, hyperactivation of inflammation after injury also promotes skeletal muscle atrophy and fibrosis. Activated AKT helps to regulate vascular function and caused vasodilation, vascular remodeling and angiogenesis[ 25 ]. In addition, AKT activation induces the expression of high levels of HIF-1, which can upregulate the expression of VEGF, thereby promoting angiogenesis[ 26 ]. This study investigated the inhibition of muscle atrophy mechanism of tuina on SNI model rats. The results showed that tuina for SNI rat recovery via regulating PI3K/Akt signaling to enhance VEGF expression. Therefore, the PI3K/Akt signaling pathway represented a promising target for exploring the pathogenesis and treatment mechanisms of PNI. Insulin-like growth factor 1 (IGF-1) is a critical hormonal regulator of muscle mass and proteostasis. IGF-1 can activate the intracellular adaptor protein insulin receptor substrate-1 and further induce downstream PI3K/Akt pathway. Moreover, IGF-1 treatment or Akt activation can negatively regulate Forkhead box O (FoxO) transcription factors, resulting in inhibition of protein degradation pathway, ubiquitin-proteasome system or autophagy-lysosome system. Evidence had shown that IGF-1 signaling cascades maintain muscle mass via suppression of FoxO-mediated autophagy and protein degradation[ 27 ]. And this study showed that IGF-1 and FoxO are major factors for tuina regulating muscle atrophy which gives us a better understanding of the mechanism of action of tuina in the treatment of PNI. However, the specific mechanisms by which tuina affects PNI remain to be elucidated. This study had limitations as it did not use the agonist or inhibitors of pathways for further verification. the current study was limited by the low number of experimental animals, future studies with large animal groups would further deepen our understanding of the effects. Moreover, we still have not cleared the mechanism of tuina for PNI. Conclusions This study revealed that tuina could effectively inhibit skeletal muscle atrophy via the microcirculation pathway in the rat model of SNI. This was accomplished by diminishing the expression levels of IGF-1, FoxO, and VEGF in the anterior tibial muscle and soleus muscle, thereby forestalling the onset and advancement of muscle atrophy. The underlying mechanism of action might be associated with the PI3K/Akt signaling pathway. Abbreviations PNI: peripheral nerve injury; SNI: sciatic nerve injury; IGF: Insulin-like growth factor 1; PI3K: phosphatidylinositol-3 kinase; FoxO: Forkhead box O; BL 37: Yinmen; GB 34: Yanglingquan; BL 57: Chengshan; MAI: Muscle atrophy index; H&E: hematoxylin and eosin staining; SFI: Sciatic function index; TFI: Tibial function index; PFI: Peroneal function index; ELISA: enzyme-linked immunosorbent assay. Declarations Acknowledgements We thank Li Wei of Beijing Jiaotong University for the technical help. Author contributions Tianyuan Yu: study conception, design of the work. Jiayue Liu, Jiawei Sun, Hongzheng Zhang, Jiawang Yan, Jinping Chen: animal experiments and data acquisition. Yue Xu, Narentuya: statistical analysis; Yingqi Zhang, Hanyu Zhang: analysis and data interpretation, drafting of the manuscript. Yingqi Zhang, Hourong Wang: approval of the final version of the manuscript. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Funding This study was supported by the National Natural Science Foundation of China (No. 82274675, 82074573) and the Beijing Natural Science Foundation (No. 7232278). Ethical approval and consent to participate The experimental designs and animal care were approved by the Ethics Committee for Animal Care and Use Committee of the Beijing University of Chinese Medicine (No. BUCM-2023032303-1119), and all procedures were conducted in strict accordance with the National Institutes of Health standards stated in the Guide for the Care and Use of Laboratory Animals. Consent for publication Not applicable. Competing interests The authors have nothing to disclose. References MOKARRAM N., DYMANUS K., SRINIVASAN A., et al. Immunoengineering nerve repair [J]. Proc Natl Acad Sci U S A, 2017, 114(26): E5077-E5084. MENORCA R. M., FUSSELL T. 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Morroniside ameliorates inflammatory skeletal muscle atrophy via inhibiting canonical and non-canonical NF-kappaB and regulating protein synthesis/degradation [J]. Front Pharmacol, 2022, 13: 1056460. CHANG C. Z., WU S. C., CHANG C. M., et al. Arctigenin, a Potent Ingredient of Arctium lappa L., Induces Endothelial Nitric Oxide Synthase and Attenuates Subarachnoid Hemorrhage-Induced Vasospasm through PI3K/Akt Pathway in a Rat Model [J]. Biomed Res Int, 2015, 2015: 490209. XU F., NA L., LI Y., et al. Roles of the PI3K/AKT/mTOR signalling pathways in neurodegenerative diseases and tumours [J]. Cell Biosci, 2020, 10(1): 54. O'NEILL B. T., LEE K. Y., KLAUS K., et al. Insulin and IGF-1 receptors regulate FoxO-mediated signaling in muscle proteostasis [J]. J Clin Invest, 2016, 126(9): 3433-3446. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 31 Dec, 2024 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted Editorial decision: Revision requested 16 Sep, 2024 Reviews received at journal 16 Sep, 2024 Reviewers agreed at journal 14 Sep, 2024 Reviewers invited by journal 01 Sep, 2024 Editor assigned by journal 01 Aug, 2024 Submission checks completed at journal 01 Aug, 2024 First submitted to journal 01 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4839192\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":338357601,\"identity\":\"a4b4970f-579b-40dd-b6a9-3ee62303db56\",\"order_by\":0,\"name\":\"英奇 张\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"英奇\",\"middleName\":\"\",\"lastName\":\"张\",\"suffix\":\"\"},{\"id\":338357604,\"identity\":\"a26da742-9c98-4b58-85e2-775e2d559ce4\",\"order_by\":1,\"name\":\"羽羽 张\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"羽羽\",\"middleName\":\"\",\"lastName\":\"张\",\"suffix\":\"\"},{\"id\":338357605,\"identity\":\"07993479-6ac5-45fd-9de4-6e96e92bfdb1\",\"order_by\":2,\"name\":\"嘉悦 刘\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"嘉悦\",\"middleName\":\"\",\"lastName\":\"刘\",\"suffix\":\"\"},{\"id\":338357606,\"identity\":\"16f2d5b5-450b-44be-b82a-69574436acb8\",\"order_by\":3,\"name\":\"佳伟 周日\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"佳伟\",\"middleName\":\"\",\"lastName\":\"周日\",\"suffix\":\"\"},{\"id\":338357607,\"identity\":\"be99f7f7-061c-43e8-9f57-279f19af7b1a\",\"order_by\":4,\"name\":\"岳 徐\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"岳\",\"middleName\":\"\",\"lastName\":\"徐\",\"suffix\":\"\"},{\"id\":338357608,\"identity\":\"ea4ddc1d-7d02-4ddb-afbe-98c6503db7a1\",\"order_by\":5,\"name\":\"Narentuya shi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Narentuya\",\"middleName\":\"\",\"lastName\":\"shi\",\"suffix\":\"\"},{\"id\":338357609,\"identity\":\"6918ad02-455d-499c-acd0-88d2ad12879e\",\"order_by\":6,\"name\":\"洪正 张\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"洪正\",\"middleName\":\"\",\"lastName\":\"张\",\"suffix\":\"\"},{\"id\":338357610,\"identity\":\"e9c4ab40-1e8e-4766-ac1a-9fe222e7fec3\",\"order_by\":7,\"name\":\"嘉旺 燕\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"嘉旺\",\"middleName\":\"\",\"lastName\":\"燕\",\"suffix\":\"\"},{\"id\":338357611,\"identity\":\"727a9541-fb36-459d-acd7-094b23a32529\",\"order_by\":8,\"name\":\"金平 陈\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"金平\",\"middleName\":\"\",\"lastName\":\"陈\",\"suffix\":\"\"},{\"id\":338357612,\"identity\":\"3cb0beb2-ca85-410d-86dc-a620cc7d9ecd\",\"order_by\":9,\"name\":\"Hourong 王\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Beijing Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hourong\",\"middleName\":\"\",\"lastName\":\"王\",\"suffix\":\"\"},{\"id\":338357613,\"identity\":\"3479aaae-dd4e-4e68-95cc-cdfa46b66cae\",\"order_by\":10,\"name\":\"天源 余\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYJCCAxCK+QBDAnEamGFa2BKI1wIFPAbEaTC4kX/wwM8dhxP7Z/d8/vBwhx0Df3s3fssMbiQzHOw9czhxxp2z2yQSzyQzSJw5u4GglgO8bYcTG27kbmNIbGNmMJDIJazl4F+glvk3ch5/SGyrJ07LYZAtG27kMEgkth0mrEXyzGODw7Jt6cYbb6SZAbUc5yHoF77jiY8/vm2zlp13I/nxx59t1XL87b34tSgcAFPNcAEevMpBQL4BTNURVDgKRsEoGAUjGAAAVtdSXPJYWewAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Beijing University of Chinese Medicine\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"天源\",\"middleName\":\"\",\"lastName\":\"余\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-08-01 04:29:11\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4839192/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4839192/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1186/s13018-024-05270-1\",\"type\":\"published\",\"date\":\"2024-12-31T15:56:58+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":63486821,\"identity\":\"93bcda23-7593-4460-b8c3-c1c7ff03f606\",\"added_by\":\"auto\",\"created_at\":\"2024-08-28 16:12:31\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":247256,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTuina improved SNI rat behavioral performance and repair of nerve injury\\u003c/p\\u003e\\n\\u003cp\\u003eA: animal experimental flow; B: Tuina intervention; C: The angle of the inclined plate changes; D: The nerve function index; D1: SFI; D2: TFI; D3: PFI. CON: control; SHA: sham; SNI: sciatic nerve injury; TUI: Tuina. Results are presented as mean ± standard deviation. \\u003csup\\u003e**\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 vs SHA; \\u003csup\\u003e##\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 vs SNI.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4839192/v1/f8ef2812d0e44a1b1735491a.png\"},{\"id\":63486819,\"identity\":\"8a131cd5-df90-4e74-88df-39d0bb2f1ecf\",\"added_by\":\"auto\",\"created_at\":\"2024-08-28 16:12:31\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":789344,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTuina alleviated SNI rat muscle atrophy\\u003c/p\\u003e\\n\\u003cp\\u003eA: ultramicrostructure of anterior tibial muscle; A1, A2, A3, A4: at low magnification; Scale bars: 1 μm; A5, A6, A7, A8: mitochondria at high magnification; Scale bars: 200 nm; A1, A5: CON group; A2, A6: SHA group; A3, A7: SNI group; A4, A8: TUI group; B: ultramicrostructure of the soleus muscle; B1, B2, B3, B4: at low magnification; Scale bars: 1 μm; B5, B6, B7, B8: mitochondria at high magnification; Scale bars: 1 μm; B1, B5: CON group; B2, B6: SHA group; B3, B7: SNI group; B4, B8: TUI group; C: MAI. C1: MAI of anterior tibial muscle; C1: MAI of soleus muscle. MAI: muscle atrophy index. Results are presented as mean ± standard deviation. \\u003csup\\u003e**\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 vs SHA; \\u003csup\\u003e##\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 vs SNI.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4839192/v1/b14778444de3014f18927bc3.png\"},{\"id\":63487331,\"identity\":\"ee7bca1b-9dfb-4b49-995b-21d61eb6f11a\",\"added_by\":\"auto\",\"created_at\":\"2024-08-28 16:20:31\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":578383,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTuina improved SNI rat microcirculation\\u003c/p\\u003e\\n\\u003cp\\u003eA: Immunofluorescence staining of sciatic nerve; Scale bars: 100 μm; A1, A2, A3, A4: DAPI staining; A5, A6, A7, A8: CD 31 staining; A9, A10, A11, A12: merge of DAPI and CD 31 staining; A1, A5, A9: CON group; A2, A6, A10: SHA group; A3, A7, A11: SNI group; A4, A8, A12: TUI group; B: Immunofluorescence staining of anterior tibial muscle; Scale bars: 100 μm; B1, B2, B3, B4: DAPI staining; B5, B6, B7, B8: CD 31 staining; B9, B10, B11, B12: merge of DAPI and CD 31 staining; B1, B5, B9: CON group; B2, B6, B10: SHA group; B3, B7, B11: SNI group; B4, B8, B12: TUI group; C: Immunofluorescence staining of soleus muscle; Scale bars: 100 μm; C1, C2, C3, C4: DAPI staining; C5, C6, C7, C8: CD 31 staining; C9, C10, C11, C12: merge of DAPI and CD 31 staining; C1, C5, C9: CON group; C2, C6, C10: SHA group; C3, C7, C11: SNI group; C4, C8, C12: TUI group. Results are presented as mean ± standard deviation. \\u003csup\\u003e*\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05 vs SHA;\\u003csup\\u003e **\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 vs SHA; \\u003csup\\u003e#\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05 vs SNI.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4839192/v1/68e5beab8ca882de44a4a843.png\"},{\"id\":63486818,\"identity\":\"86321dad-eb39-47d4-b40d-710ee7174c42\",\"added_by\":\"auto\",\"created_at\":\"2024-08-28 16:12:31\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":67286,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTuina regulated muscle atrophy associated with IGF-1 and FoxO\\u003c/p\\u003e\\n\\u003cp\\u003eA: Relative protein expression of the anterior tibial muscle; A1: IGF-1; A2: FoxO; B: Relative protein expression of the soleus muscle; B1: IGF-1; B2: FoxO; Results are presented as mean ± standard deviation.\\u003csup\\u003e **\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 vs SHA; \\u003csup\\u003e#\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05 vs SNI; \\u003csup\\u003e##\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 vs SNI.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4839192/v1/b77690fd07e8e8251b6db718.png\"},{\"id\":63486822,\"identity\":\"7841fa05-aeb1-47a6-a1e7-b21cf2038e74\",\"added_by\":\"auto\",\"created_at\":\"2024-08-28 16:12:31\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":223776,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTuina regulated factors associated with the PI3K/AKT pathway\\u003c/p\\u003e\\n\\u003cp\\u003eA: Relative protein expression of the anterior tibial muscle; A1: p-PI3K, p-AKT; A2: VEGF; B: Quantification of specific signal intensities. B1: p-PI3K; B2: p-AKT; B3: VEGF; C: Relative protein expression of the soleus muscle; C1: p-PI3K, p-AKT; C2: VEGF; D: Quantification of specific signal intensities. D1: p-PI3K; D2: p-AKT; D3: VEGF; Results are presented as mean ± standard deviation.\\u003csup\\u003e *\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05 vs SHA; \\u003csup\\u003e**\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 v\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp; s SHA; \\u003csup\\u003e#\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05 vs SNI; \\u003csup\\u003e##\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 vs SNI.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4839192/v1/bcfabf84da1ec2b807767efd.png\"},{\"id\":73093219,\"identity\":\"b97ba58f-6ec0-4475-b46f-7d61b44d826c\",\"added_by\":\"auto\",\"created_at\":\"2025-01-06 16:11:01\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2465749,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4839192/v1/d0980ee8-9ad5-4682-bbc6-510509986b16.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Tuina alleviates the muscle atrophy of sciatic nerve injury rats through regulating PI3K/Akt signaling\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003ePeripheral nerve injury (PNI), a neurological condition that seriously threatens both human and animal patients[\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e], a big percentage of which is associated with poor functional outcomes, insufficient nerve recovery, and the loss of motor function. These are followed by partial recovery, muscle atrophy, and profound weakness[\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. The change of microvascular after nerve injury is an important reason for muscle atrophy. Injury-induced loss of local blood vessels contribute to inflammation and ischemia and thus to the overall damage to the nerve and even muscle that innervate[\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. The reversal of microvascular dysfunction may provide new approaches for the treatment and prevention of PNI diseases.\\u003c/p\\u003e \\u003cp\\u003eSkeletal muscle atrophy is a debilitating consequence of denervation, of which is one of the contributing factors towards incomplete functional recovery[\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. A combination of factors, including increased proteolysis, decreased protein synthesis, and impaired regenerative capacity, contributes to skeletal muscle atrophy [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. The activation of phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway prevents muscle atrophy through inhibiting the activity of FoxO transcription factors and augmenting protein synthesis. PI3K/Akt signaling can also dominantly inhibit the effects of myostatin causing an increase in skeletal muscle size[\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Moreover, PI3K, as well as its downstream target Akt, has been implicated in a number of cellular responses linked to angiogenesis, including endothelial cell migration and survival. Overexpression of constitutively active PI3K or Akt promotes angiogenesis in vivo and increases VEGF expression[\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTuina, as a type of non-surgical interventions, is an alternative medical therapy which is safe and has virtually no side effects. Accumulating literature have reported its application in multiple diseases improving disability and pain in patients[\\u003cspan additionalcitationids=\\\"CR9\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Additionally, due to its anti-inflammatory and blood circulation-promoting effects, Tuina manipulations are widely used to myopathy recovery[\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Studies have confirmed that Tuina has a good effect on the treatment of PNI through autophagy, synaptic plasticity, axon regeneration, and remyelination, and ultimately achieve the purpose of restoring sensory and motor function[\\u003cspan additionalcitationids=\\\"CR13\\\" citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, whether the mechanical effect of tuina on PNI inhibit skeletal muscle atrophy via the microcirculation pathway remains unknown. Additionally, whether this process is regulated by the PI3K/AKT pathway deserves further exploration. To address these questions, we established a rat model of SNI and observed the effects of tuina on muscle function, muscle atrophy, microcirculation and PI3K/AKT pathway.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnimals\\u003c/h2\\u003e \\u003cp\\u003e The animal protocol was approved by the Animal Care and Use Committee of the Beijing University of Chinese Medicine (No. BUCM-2023032303-1119). All animal experiments were designed by the principles of the 3Rs (Replacement, Reduction and Refinement) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague\\u0026ndash;Dawley (SD) rats (6-week-old, 190\\u0026ndash;210 g) were purchased from Beijing SPF Biotechnology Co., Ltd. (Beijing, China), and dwelled in a pathogen-free environment with 4 animals per cage. The rats are fed in a 12-h light-dark cycle environment, temperature (25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.5) ℃, humidity 40% \\u0026minus;\\u0026thinsp;50%, and have free access to diet and drinking water.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSNI model\\u003c/h2\\u003e \\u003cp\\u003e36 rats were randomly divided into four groups (n\\u0026thinsp;=\\u0026thinsp;9): Blank Control (CON) group, Sham Model (SHA) group, SNI Model (SNI) group and Tuina (TUI) group. Pathological modeling started after one week of acclimatization. The rat model of SNI was established as previously described[\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. After anesthetized, an incision of about 1 cm long was made in the skin along the direction of the sciatic nerve exposing the lower edge of the piriformis. For the rats in SNI and TUI groups, the special hemostatic pliers were used to clamp 5 mm at the distal end of the sciatic nerve nodule for 5 s with full force (6 N) resulting in a length of about 2 mm injury point. For the rats in SHA group, the sciatic nerve was only found, sterilized, sutured, and then sterilized.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTuina intervention\\u003c/h2\\u003e \\u003cp\\u003eThe TUI group received \\u0026ldquo;Three-Manipulation and Three-Acupoint\\u0026rdquo; treatment, the procedure was performed as follows: The Tuina Manipulation Simulator (Self-developed machine, China invention patent number ZL202320511277.5) was set to stimulate with a force of 4 N, 60 times per minute (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). The stimulus rod was placed on BL 37, GB 34, and BL 57 of the surgical side, then finger pressing, plucking, and kneading manipulation were stimulated, respectively. Each acupoint and manipulation were operated for 1 min consecutively for 9 min in total. There were ten times treatments following 1 day rest and ten times treatments were repeated, so that a total of 20 times treatments were completed.\\u003c/p\\u003e \\u003cp\\u003eThe grip restraint intervention was performed in the SHA and SNI groups. In order to reduce animal stress response, petting and stroking animals for 9 min before the formal intervention every day.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBehavioral assessment\\u003c/h2\\u003e \\u003cp\\u003eAn electrically inclined plate tester was used to detect the changes in the muscle strength of the rats\\u0026rsquo; hind limbs. Rats in each group were taken for behavioral testing and the test was measured at baseline before the surgery, on the day of 10-time and 20-time after the intervention. The heads of the rats were placed on the board toward the end and gradually increase the angle of the board after the rats calmed down. When the rats cannot stay in this position for 5 s, the critical angle of the protractor was recorded, and the average level of three measurements was taken.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eNerve function index\\u003c/h2\\u003e \\u003cp\\u003eNerve function index were collected using the DigiGait\\u0026trade; Imaging System and then analyzed by the DigiGait\\u0026trade; 15.0 analysis software (Mouse Specifics, Inc.; Quincy, MA, USA). The rats were habituated to the apparatus 1 week before the experiment, with the treadmill belt gradually accelerated to 10 cm/s.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMuscle atrophy index (MAI)\\u003c/h2\\u003e \\u003cp\\u003eAfter the rats were sacrificed, the anterior tibial muscle and soleus muscle were removed, and their wet weights were measured using an electronic balance. MAI is defined by muscle weight divided by body weight. The empty stomach weights of the rat pre-dissection and their anterior tibial muscle and soleus muscle (post-dissection) were measured.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTransmission electron microscopy observation\\u003c/h2\\u003e \\u003cp\\u003eThe anterior tibial muscle, soleus muscle and sciatic nerve were removed, fixed in pre-cooled 2.5% (w/v) glutaraldehyde for 3 h, 0.1M PB washed. Then were postfixed in a 1% (w/v) osmic acid solution for 1 h, 0.1M PB washed for 1h, dehydrated (through a grade series of ethanol solutions) and embedded in Epon 812 epoxy resin. The segments were cut into 70nm-thick ultrathin slices. Then the sections were stained with saturated aqueous uranyl acetate (2%) and citrate and were observed and analyzed by TEM.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunofluorescence\\u003c/h2\\u003e \\u003cp\\u003eThe anterior tibial muscle, soleus muscle and sciatic nerve were fixed in 4% paraformaldehyde at 4 ◦C, dehydrated using a sucrose gradient, embedded in optimal cutting temperature compound, and cut into 4um sections. Paraffin sections were dewaxed, dehydrated, subjected to antigen retrieval, cleared of spontaneous fluorescence, and blocked with serum. Primary antibodies for CD 31(1:500, abcam, USA) were given for incubation at 4 ℃ overnight. Sections were rinsed with phosphate buffered saline and incubated with appropriate secondary antibodies at 37℃ for 30min, then rinsed with phosphate buffered saline. The sections were observed under a fluorescence microscope, and Image J was used to analyze the Pearson coefficient of immunofluorescence colocalization and the fluorescence intensity of each protein.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEnzyme-linked immunosorbent assay (ELISA)\\u003c/h2\\u003e \\u003cp\\u003e50 milligrams of the gastrocnemius muscle and tibialis anterior muscle were weighed, and PBS was added at a weight (mg)/volume (\\u0026micro;L) ratio of 1:10 for homogenization. protein concentrations of the samples were determined with the ELISA kit. The test procedures were as follows: standard dilution, sample addition, washing, color development, reaction termination, then the absorbance of each well was measured, and finally calculated the concentration of FoxO, and IGF-1 by drawing a standard curve.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern blotting\\u003c/h2\\u003e \\u003cp\\u003eThe gastrocnemius muscle, tibialis anterior muscle and sciatic nerve were lysed in RIPA lysis buffer. The BCA protein assay kit was used to measure the protein concentration. Briefly, 20 \\u0026micro;g of total protein was separated by SDS-PAGE and transferred to PVDF membranes at 400 mA for 30 min. Subsequently, the membranes were blocked for 30min at room temperature with Blocking solution. The membranes were incubated overnight at 4\\u0026deg;C with the following primary antibodies: mouse anti-GAPDH (1:10000, Ym3029, Immunoway), rabbit anti-p-PI3K (1:1000, AF3241, Affbiotech), rabbit anti-p-Akt (1:1000, bs-0867R, Bioss), and rabbit anti-VEGF (1:1000, bs-1665R, Bioss). After washing three times with TBST, the secondary HRP-conjugated antibodies were as follows: goat anti-rabbit IgG H\\u0026amp;L (1:10000, bs-0295G, Bioss), goat anti-mouse IgG H\\u0026amp;L (1:10000, bs-0296G, Bioss). After washing three times with TBST, an ECL kit was used to detect the immunoactivity.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eData analysis was performed with SPSS Statistics software Version 26.0 (IBM). Results were presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD. Student\\u0026rsquo;s t-test was used to compare differences between two groups. One-way ANOVA was used for comparisons between groups, and the LSD multiple comparison test was used for multiple comparisons. \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was treated as statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec15\\\"\\u003e\\n \\u003ch2\\u003eTuina improved behavioral performance\\u003c/h2\\u003e\\n \\u003cp\\u003eThere was no redness or swelling point of injury for rats in each group, and the health status was good after the modeling operation. After the SNI model was established, the fine motor function was greatly affected, indicating that the model was successfully prepared.\\u003c/p\\u003e\\n \\u003cp\\u003eFor behavioral assessment, the angle of the inclined plate was used to evaluate the muscle strength and motor function. And the results showed that the angle of the inclined plate of the rats in the CON and SHA group before intervention, after 10-time and 20-time interventions were not statistically difference. Compared with the SHA group, the inclined plate angle of the rats in the SNI group and the TUI group decreased significantly before intervention but there was no significant difference between the SNI group and the TUI group. Compared with those in the SNI group after the 10-time and 20-time interventions, the inclined plate angle of the rats in the TUI group increased significantly, but there were still significant differences compared with those in the SHA group. Those results showed that tuina intervention can effectively improve the motor function in the lower limbs after nerve injury (Fig. \\u003cspan\\u003e1\\u003c/span\\u003eC).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec16\\\"\\u003e\\n \\u003ch2\\u003eTuina promoted the repair of nerve injury\\u003c/h2\\u003e\\n \\u003cp\\u003eThe nerve function index was used to evaluate the recovery of injury-nerve. The results of rats in each group showed that, compared with the baseline, the Sciatic function index (SFI), Tibial function index (TFI), and Peroneal function index (PFI) of rats in the SNI group and the TUI group decreased significantly before intervention; compared with the SNI group after the 10-time and 20-time interventions, the SFI, TFI, PFI of rats in the TUI group increased significantly. Those results showed that tuina intervention can effectively promote the recovery of fine movements in the hind limbs of rats and facilitate the recovery of motor function of nerve injury (Fig. \\u003cspan\\u003e1\\u003c/span\\u003eD).\\u003c/p\\u003e\\n \\u003cp\\u003eA: animal experimental flow; B: Tuina intervention; C: The angle of the inclined plate changes; D: The nerve function index; D1: SFI; D2: TFI; D3: PFI. CON: control; SHA: sham; SNI: sciatic nerve injury; TUI: Tuina. Results are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation. \\u003csup\\u003e**\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 vs SHA; \\u003csup\\u003e##\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 vs SNI.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec17\\\"\\u003e\\n \\u003ch2\\u003eTuina alleviated muscle atrophy\\u003c/h2\\u003e\\n \\u003cp\\u003eMuscle atrophy index (MAI) was used to evaluate the muscle atrophy. The MAI of the anterior tibial muscle and soleus muscle had the similar changes among four groups. Results of the rats in each group showed that, there were no statistically differences in the CON and SHA group. Compared with the SHA group, the MAI of the rats in the SNI group and the TUI group decreased significantly. Compared with the SNI group, the TUI group increased significantly, but there were still significant differences compared with those in the SHA group. Those results showed that tuina intervention can effectively relieve muscle atrophy caused by nerve damage (Fig. \\u003cspan\\u003e2\\u003c/span\\u003eC).\\u003c/p\\u003e\\n \\u003cp\\u003eTransmission electron microscopy observation was used to evaluate the ultrastructural changes of muscles. Results of the anterior tibial muscle and soleus muscle showed that, in the CON and SHA groups: the myosin filaments were arranged neatly and the transverse lines were obvious. A band was formed by interlacing and overlapping heterotropic myosin filaments and actin filaments. Only the actin filaments pass through the region, showing an isotropic I band; there were regular Z lines between I band; obvious sarcomeres between the Z line junctions. The M line is the site of marked thickening of myosin. A large number of mitochondria were present among the muscle fibers, mitochondrial morphology normal and regular. In the SNI group: the myofilaments were arranged disorderly and the horizontal lines disappeared. Typical structures of skeletal muscle such as A band, I band, Z line, M line disappeared. The space between muscle bundles was significantly enlarged. A large number of vacuolated mitochondria were found between muscle bundles and muscle fibers. In the TUI group: the myofilaments were arranged neatly and horizontal lines appeared. Clearly visible lines to Z line and M line, A band and I band could identify. The internal structure of mitochondria between myofilaments and fascicles was not clear, and occasionally vacuolated mitochondria were observed. Those results showed that tuina intervention can effectively relieve ultrastructural changes of muscle atrophy caused by nerve damage (Fig.\\u0026nbsp;2AB)\\u003c/p\\u003e\\n \\u003cp\\u003eA: ultramicrostructure of anterior tibial muscle; A1, A2, A3, A4: at low magnification; Scale bars: 1 \\u0026micro;m; A5, A6, A7, A8: mitochondria at high magnification; Scale bars: 200 nm; A1, A5: CON group; A2, A6: SHA group; A3, A7: SNI group; A4, A8: TUI group; B: ultramicrostructure of the soleus muscle; B1, B2, B3, B4: at low magnification; Scale bars: 1 \\u0026micro;m; B5, B6, B7, B8: mitochondria at high magnification; Scale bars: 1 \\u0026micro;m; B1, B5: CON group; B2, B6: SHA group; B3, B7: SNI group; B4, B8: TUI group; C: MAI. C1: MAI of anterior tibial muscle; C1: MAI of soleus muscle. MAI: muscle atrophy index. Results are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation. \\u003csup\\u003e**\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 vs SHA; \\u003csup\\u003e##\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 vs SNI.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec18\\\"\\u003e\\n \\u003ch2\\u003eTuina enhanced microcirculation\\u003c/h2\\u003e\\n \\u003cp\\u003eImmunofluorescence was used to evaluate the microcirculation of muscles and nerve. Figure \\u003cspan\\u003e3\\u003c/span\\u003e shows the results of immunofluorescence staining for CD 31 from each group. The nuclei were stained blue with DAPI, and CD 31 was stained red and marked microvascular endothelial cells. The expression of CD 31 in sciatic nerve, anterior tibial muscle and soleus muscle had the similar changes among four groups. Compared with CON and SHA group, the expression of CD 31 in the SNI group was significantly reduced. Compared with that in the SHA group, the fluorescence intensity of CD 31 in the SNI group was significantly decreased. A CD 31 distribution was observed in the TUI group, and the fluorescence intensity was significantly higher in the TUI group than in the SNI group. Those results showed that tuina intervention can effectively increase microvascular density after nerve injury in rats (Fig. 3ABC).\\u003c/p\\u003e\\n \\u003cp\\u003eA: Immunofluorescence staining of sciatic nerve; Scale bars: 100 \\u0026micro;m; A1, A2, A3, A4: DAPI staining; A5, A6, A7, A8: CD 31 staining; A9, A10, A11, A12: merge of DAPI and CD 31 staining; A1, A5, A9: CON group; A2, A6, A10: SHA group; A3, A7, A11: SNI group; A4, A8, A12: TUI group; B: Immunofluorescence staining of anterior tibial muscle; Scale bars: 100 \\u0026micro;m; B1, B2, B3, B4: DAPI staining; B5, B6, B7, B8: CD 31 staining; B9, B10, B11, B12: merge of DAPI and CD 31 staining; B1, B5, B9: CON group; B2, B6, B10: SHA group; B3, B7, B11: SNI group; B4, B8, B12: TUI group; C: Immunofluorescence staining of soleus muscle; Scale bars: 100 \\u0026micro;m; C1, C2, C3, C4: DAPI staining; C5, C6, C7, C8: CD 31 staining; C9, C10, C11, C12: merge of DAPI and CD 31 staining; C1, C5, C9: CON group; C2, C6, C10: SHA group; C3, C7, C11: SNI group; C4, C8, C12: TUI group. Results are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation. \\u003csup\\u003e*\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 vs SHA; \\u003csup\\u003e**\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 vs SHA; \\u003csup\\u003e#\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 vs SNI.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec19\\\"\\u003e\\n \\u003ch2\\u003eTuina regulated muscle atrophy associated with IGF-1 and FoxO\\u003c/h2\\u003e\\n \\u003cp\\u003eAs shown in Fig. \\u003cspan\\u003e4\\u003c/span\\u003e, the anterior tibial muscle and soleus muscle expression levels of IGF-1were significantly lower in the SNI group and TUI group compared with the SHA group. Conversely, the TUI group displayed an increase in the expression levels of IGF-1compared with the SNI group. The expression levels of FoXO were significantly higher in the SNI group compared with the SHA group. The TUI group displayed a decrease in the expression levels of FoXO compared with the SNI group. These outcomes suggested that tuina could alleviate the muscle atrophy after SNI associated with IGF-1 and FoxO (Fig.\\u0026nbsp;4AB).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec20\\\"\\u003e\\n \\u003ch2\\u003eTuina regulated factors associated with the PI3K/AKT pathway\\u003c/h2\\u003e\\n \\u003cp\\u003eWestern blotting was used to evaluate the changes of anterior tibial muscle and soleus muscle in the PI3K/AKT signaling pathway. The results revealed that the protein expression levels of p-PI3K and p-AKT significantly decreased in the SNI group. However, tuina intervention enhanced the expression. The protein expression levels of VEGF significantly increased in the TUI group, comparing with SNI. This suggested that tuins had a certain effect on the PI3K/AKT signaling pathway in the anterior tibial muscle and soleus muscle of rats with SNI (Fig.\\u0026nbsp;5ABCD)\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eTuina, a non-pharmacological therapy with little or no side effects, has been extensively used in clinical practice for the relief and treatment of diseases. Under the guidance of traditional chinese medicine and western medicine anatomy and pathology, tuina acts on the body surface by various manipulations to regulate the physiological and pathological state, so as to treat diseases. Studies have shown that the benefits of tuina therapy include increased blood flow, reduced pain and perceived fatigue, attenuated inflammatory signals of muscle injury, improved muscle strength and self-perception of muscle injury, reduced muscle tension[\\u003cspan additionalcitationids=\\\"CR16\\\" citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Tuina has advantages of not causing dependence, easier use, faster effect onset, and increased popularity among worldwide people including athletes. Related studies involve case reports, meta-analyses, controlled clinical trials, and animal experiments showed its positive effect on all kinds of injury. In this study, we found that tuina intervention could improve SNI rats hindlimb motor function by promoting nerve repair and inhibiting muscle atrophy.\\u003c/p\\u003e \\u003cp\\u003eSNI model was used for simulating clinical PNI, which allows the evaluation of neuropathic changes and nerve regeneration. The \\u0026ldquo;Three-Manipulation and Three-Acupoint\\u0026rdquo; is a combination of manipulations and acupoints that we have studied and proven to be effective. \\u003cem\\u003eYinmen\\u003c/em\\u003e (BL 37) is located in the body surface projection area of the sciatic nerve trunk, at the location of biceps femoris muscle; \\u003cem\\u003eChengshan\\u003c/em\\u003e (BL 57) is located in the body surface projection area of the tibial nerve, at the location of gastrocnemius muscle and \\u003cem\\u003eYanglingquan\\u003c/em\\u003e (GB 34) is located in the body surface projection area of the common peroneal nerve, at the location of tibialis anterior muscle. We found that after 20-time intervention, the function and structure of muscles and nerve in the intervention area were significantly improved, and demonstrated significant effects of the \\u0026ldquo;Three-Manipulation and Three-Acupoint\\u0026rdquo;.\\u003c/p\\u003e \\u003cp\\u003eSciatic damage was estimated to contribute to 90% inhibition of muscle mass. Moreover, soleus muscles retained only 8% contractibility in 14-week denervated rats, the tibial anterior experienced 3\\u0026ndash;5% contraction in long-term denervation [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. Evidence had shown that tuina could effectively accelerate the recovery of muscle fibers, improve the structure and morphology of gastrocnemius muscle, and delay the atrophy after sciatic nerve transection[\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. In this study, we found that SNI model led to the mass of anterior tibial muscle and soleus muscle decrease and ultrastructure change, and after tuina intervention, the mass of muscles increased significantly and ultrastructure restored obviously.\\u003c/p\\u003e \\u003cp\\u003eMitochondria play important roles in maintaining cellular homeostasis and skeletal muscle health, mitochondrial dysfunction can lead to skeletal muscle atrophy. During muscle atrophy, mitochondrial degradation influences the reduction of mitochondrial quality and quantity, which is controlled by mitochondrial autophagy as well as mitochondrial fusion and fission kinetics[\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Therefore, in the strategy of treating muscle atrophy diseases, mitochondrial is the key entry point. Crane et al. showed that tuina therapy appears to be clinically beneficial by promoting mitochondrial biogenesis[\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. In this study, the results of electron microscope showed that the structure and morphology of mitochondrial is better in SNI rats treated with tuina than SNI model rats.\\u003c/p\\u003e \\u003cp\\u003eThe PI3K/Akt pathway is one of the most important pathways that regulates muscle atrophy. Muscle atrophy usually leads to the loss of muscle mass and function and is characterized by a reduction in muscle fiber size and mass, and an imbalance between protein synthesis and degradation in the muscle. Protein synthesis in skeletal muscle is mainly regulated by the PI3K/Akt signaling axis. This pathway plays a critical role in myotube hypertrophy, and activation of Akt in rat muscle prevents denervation-induced atrophy[\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. Additionally, an increase in the levels of the PI3K/Akt signaling pathway is critical for autophagy suppression, the formation of autophagosomes is stimulated by decreased levels of PI3K[\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Skeletal muscle loses its contractile function after denervation, resulting in reduced blood perfusion and, thus, leading to nutritional and metabolic abnormalities in the target muscle, hyperactivation of inflammation after injury also promotes skeletal muscle atrophy and fibrosis. Activated AKT helps to regulate vascular function and caused vasodilation, vascular remodeling and angiogenesis[\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. In addition, AKT activation induces the expression of high levels of HIF-1, which can upregulate the expression of VEGF, thereby promoting angiogenesis[\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. This study investigated the inhibition of muscle atrophy mechanism of tuina on SNI model rats. The results showed that tuina for SNI rat recovery via regulating PI3K/Akt signaling to enhance VEGF expression. Therefore, the PI3K/Akt signaling pathway represented a promising target for exploring the pathogenesis and treatment mechanisms of PNI.\\u003c/p\\u003e \\u003cp\\u003eInsulin-like growth factor 1 (IGF-1) is a critical hormonal regulator of muscle mass and proteostasis. IGF-1 can activate the intracellular adaptor protein insulin receptor substrate-1 and further induce downstream PI3K/Akt pathway. Moreover, IGF-1 treatment or Akt activation can negatively regulate Forkhead box O (FoxO) transcription factors, resulting in inhibition of protein degradation pathway, ubiquitin-proteasome system or autophagy-lysosome system. Evidence had shown that IGF-1 signaling cascades maintain muscle mass via suppression of FoxO-mediated autophagy and protein degradation[\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. And this study showed that IGF-1 and FoxO are major factors for tuina regulating muscle atrophy which gives us a better understanding of the mechanism of action of tuina in the treatment of PNI. However, the specific mechanisms by which tuina affects PNI remain to be elucidated. This study had limitations as it did not use the agonist or inhibitors of pathways for further verification. the current study was limited by the low number of experimental animals, future studies with large animal groups would further deepen our understanding of the effects. Moreover, we still have not cleared the mechanism of tuina for PNI.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eThis study revealed that tuina could effectively inhibit skeletal muscle atrophy via the microcirculation pathway in the rat model of SNI. This was accomplished by diminishing the expression levels of IGF-1, FoxO, and VEGF in the anterior tibial muscle and soleus muscle, thereby forestalling the onset and advancement of muscle atrophy. The underlying mechanism of action might be associated with the PI3K/Akt signaling pathway.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003ePNI: peripheral nerve injury; SNI: sciatic nerve injury; IGF: Insulin-like growth factor 1; PI3K: phosphatidylinositol-3 kinase; FoxO: Forkhead box O; BL 37: Yinmen; GB 34: Yanglingquan; BL 57: Chengshan; MAI: Muscle atrophy index; H\\u0026amp;E: hematoxylin and eosin staining; SFI: Sciatic function index; TFI: Tibial function index; PFI: Peroneal function index; ELISA: enzyme-linked immunosorbent assay.\\u0026nbsp;\\u003c/p\\u003e\\n\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank Li Wei of Beijing Jiaotong University for the technical help.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTianyuan Yu: study conception, design of the work.\\u0026nbsp;Jiayue Liu, Jiawei Sun, Hongzheng Zhang, Jiawang Yan, Jinping Chen: animal experiments and data acquisition. Yue Xu, Narentuya: statistical analysis;\\u0026nbsp;Yingqi Zhang, Hanyu Zhang: analysis and data interpretation, drafting of the manuscript. Yingqi Zhang, Hourong Wang: approval of the final version of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was supported by the National Natural Science Foundation of China (No. 82274675, 82074573) and the Beijing Natural Science Foundation (No. 7232278).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical approval and consent to participate\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe experimental designs and animal care were approved by the Ethics Committee for Animal Care and Use Committee of the Beijing University of Chinese Medicine (No. BUCM-2023032303-1119), and all procedures were conducted in strict accordance with the National Institutes of Health standards stated in the Guide for the Care and Use of Laboratory Animals.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors have nothing to disclose.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eMOKARRAM N., DYMANUS K., SRINIVASAN A., et al. Immunoengineering nerve repair [J]. Proc Natl Acad Sci U S A, 2017, 114(26): E5077-E5084.\\u003c/li\\u003e\\n\\u003cli\\u003eMENORCA R. M., FUSSELL T. S., ELFAR J. C. Nerve physiology: mechanisms of injury and recovery [J]. Hand Clin, 2013, 29(3): 317-330.\\u003c/li\\u003e\\n\\u003cli\\u003eOUDEGA M. Molecular and cellular mechanisms underlying the role of blood vessels in spinal cord injury and repair [J]. Cell Tissue Res, 2012, 349(1): 269-288.\\u003c/li\\u003e\\n\\u003cli\\u003eZAINUL Z., HEIKKINEN A., KOIVISTO H., et al. Collagen XIII Is Required for Neuromuscular Synapse Regeneration and Functional Recovery after Peripheral Nerve Injury [J]. J Neurosci, 2018, 38(17): 4243-4258.\\u003c/li\\u003e\\n\\u003cli\\u003eZHANG H., QI G., WANG K., et al. Oxidative stress: Roles in skeletal muscle atrophy [J]. Biochem Pharmacol, 2023, 214: 115664.\\u003c/li\\u003e\\n\\u003cli\\u003eGLASS D. J. PI3 kinase regulation of skeletal muscle hypertrophy and atrophy [J]. Curr Top Microbiol Immunol, 2010, 346: 267-278.\\u003c/li\\u003e\\n\\u003cli\\u003eJIA L., ZHENG P., WANG H., et al. VEGF alleviates lower limb ischemia in diabetic mice by altering muscle fiber types [J]. Exp Ther Med, 2022, 23(4): 251.\\u003c/li\\u003e\\n\\u003cli\\u003eZHOU X., KONG L., REN J., et al. Effect of traditional Chinese exercise combined with massage on pain and disability in patients with lumbar disc herniation: A multi-center, randomized, controlled, assessor-blinded clinical trial [J]. Front Neurol, 2022, 13: 952346.\\u003c/li\\u003e\\n\\u003cli\\u003eCABANAS-VALDES R., CALVO-SANZ J., SERRA-LLOBET P., et al. The Effectiveness of Massage Therapy for Improving Sequelae in Post-Stroke Survivors. A Systematic Review and Meta-Analysis [J]. Int J Environ Res Public Health, 2021, 18(9).\\u003c/li\\u003e\\n\\u003cli\\u003eLIU Z. F., ZHANG Y., LIU J., et al. Effect of Traditional Chinese Non-Pharmacological Therapies on Knee Osteoarthritis: A Narrative Review of Clinical Application and Mechanism [J]. Orthop Res Rev, 2024, 16: 21-33.\\u003c/li\\u003e\\n\\u003cli\\u003eHUANG B., RUAN L., WANG L., et al. Mimicking Ding\\u0026apos;s Roll Method on Notexin-Induced Muscle Injury in Rats [J]. J Vis Exp, 2023, (198).\\u003c/li\\u003e\\n\\u003cli\\u003eLIU Z., WANG H., YU T., et al. A Review on the Mechanism of Tuina Promoting the Recovery of Peripheral Nerve Injury [J]. Evid Based Complement Alternat Med, 2021, 2021: 6652099.\\u003c/li\\u003e\\n\\u003cli\\u003eLV T. T., MO Y. J., YU T. Y., et al. Using RNA-Seq to Explore the Repair Mechanism of the Three Methods and Three-Acupoint Technique on DRGs in Sciatic Nerve Injured Rats [J]. Pain Res Manag, 2020, 2020: 7531409.\\u003c/li\\u003e\\n\\u003cli\\u003eLYU T., LIU Z., YU T., et al. Applying RNA sequencing technology to explore repair mechanism of Tuina on gastrocnemius muscle in sciatic nerve injury rats [J]. Chin Med J (Engl), 2022, 135(19): 2378-2379.\\u003c/li\\u003e\\n\\u003cli\\u003eNUNES G. S., BENDER P. U., DE MENEZES F. S., et al. Massage therapy decreases pain and perceived fatigue after long-distance Ironman triathlon: a randomised trial [J]. J Physiother, 2016, 62(2): 83-87.\\u003c/li\\u003e\\n\\u003cli\\u003eSHIN M. S., SUNG Y. H. Effects of Massage on Muscular Strength and Proprioception After Exercise-Induced Muscle Damage [J]. J Strength Cond Res, 2015, 29(8): 2255-2260.\\u003c/li\\u003e\\n\\u003cli\\u003eKANG L., LIU P., PENG A., et al. Application of traditional Chinese therapy in sports medicine [J]. Sports Med Health Sci, 2021, 3(1): 11-20.\\u003c/li\\u003e\\n\\u003cli\\u003eYADAV A., DABUR R. Skeletal muscle atrophy after sciatic nerve damage: Mechanistic insights [J]. Eur J Pharmacol, 2024, 970: 176506.\\u003c/li\\u003e\\n\\u003cli\\u003eMA S. J., ZHANG J. P., HUA X. Y., et al. Tuina therapy promotes behavioral improvement and brain plasticity in rats with peripheral nerve injury and repair [J]. Brain Behav, 2023, 13(9): e3174.\\u003c/li\\u003e\\n\\u003cli\\u003ePAN F., YU T. Y., WONG S., et al. Chinese tuina downregulates the elevated levels of tissue plasminogen activator in sciatic nerve injured Sprague-Dawley rats [J]. Chin J Integr Med, 2017, 23(8): 617-624.\\u003c/li\\u003e\\n\\u003cli\\u003eCHEN X., JI Y., LIU R., et al. Mitochondrial dysfunction: roles in skeletal muscle atrophy [J]. J Transl Med, 2023, 21(1): 503.\\u003c/li\\u003e\\n\\u003cli\\u003eCRANE J. D., OGBORN D. I., CUPIDO C., et al. Massage therapy attenuates inflammatory signaling after exercise-induced muscle damage [J]. Sci Transl Med, 2012, 4(119): 119ra113.\\u003c/li\\u003e\\n\\u003cli\\u003eBONALDO P., SANDRI M. Cellular and molecular mechanisms of muscle atrophy [J]. Dis Model Mech, 2013, 6(1): 25-39.\\u003c/li\\u003e\\n\\u003cli\\u003eYI X., TAO J., QIAN Y., et al. Morroniside ameliorates inflammatory skeletal muscle atrophy via inhibiting canonical and non-canonical NF-kappaB and regulating protein synthesis/degradation [J]. Front Pharmacol, 2022, 13: 1056460.\\u003c/li\\u003e\\n\\u003cli\\u003eCHANG C. Z., WU S. C., CHANG C. M., et al. Arctigenin, a Potent Ingredient of Arctium lappa L., Induces Endothelial Nitric Oxide Synthase and Attenuates Subarachnoid Hemorrhage-Induced Vasospasm through PI3K/Akt Pathway in a Rat Model [J]. Biomed Res Int, 2015, 2015: 490209.\\u003c/li\\u003e\\n\\u003cli\\u003eXU F., NA L., LI Y., et al. Roles of the PI3K/AKT/mTOR signalling pathways in neurodegenerative diseases and tumours [J]. Cell Biosci, 2020, 10(1): 54.\\u003c/li\\u003e\\n\\u003cli\\u003eO\\u0026apos;NEILL B. T., LEE K. Y., KLAUS K., et al. Insulin and IGF-1 receptors regulate FoxO-mediated signaling in muscle proteostasis [J]. J Clin Invest, 2016, 126(9): 3433-3446.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-orthopaedic-surgery-and-research\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"josr\",\"sideBox\":\"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)\",\"snPcode\":\"13018\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13018/3\",\"title\":\"Journal of Orthopaedic Surgery and Research\",\"twitterHandle\":\"@MSKmedBMC\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Peripheral nerve injury, Tuina, Muscle atrophy, Microcirculation, PI3K/AKT pathway\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4839192/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4839192/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eTuina has been shown to be an effective treatment for the decrease of skeletal muscle atrophy after Peripheral nerve injury (PNI). However, its mechanism is unclear. This study aimed to explore the underlying mechanisms of tuina on rats with sciatic nerve injury (SNI).\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e \\u003cp\\u003eA rat model of SNI was established. After a total of 20 times tuina intervention, the curative effects were evaluated by behavioral assessment, nerve function index and muscle atrophy index (MAI). The pathological changes were observed by transmission electron microscopy and immunofluorescence. The levels of IGF-1 and FoxO were detected by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to detect the expression of proteins in the PI3K/AKT signaling pathway.\\u003c/p\\u003e\\u003ch2\\u003eResult\\u003c/h2\\u003e \\u003cp\\u003eThe behavioral assessment, nerve function index and MAI proved that tuina significantly improved muscle atrophy after SNI compared with that in SNI model group. Transmission electron microscopy showed that tuina improved ultramicrostructure and immunofluorescence of CD31 showed that tuina improved microcirculation. Further, we observed that tuina differentially regulated levels of IGF-1 and FoxO and the protein expression of p-PI3K, p-AKT and VEGF in anterior tibial muscle and soleus muscle.\\u003c/p\\u003e\\u003ch2\\u003eConclusion\\u003c/h2\\u003e \\u003cp\\u003eTuina could effectively inhibit skeletal muscle atrophy via the microcirculation pathway in the rat model of SNI, by regulating the expression levels of IGF-1, FoxO. The underlying mechanism of action might be via the PI3K/Akt signaling pathway.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Tuina alleviates the muscle atrophy of sciatic nerve injury rats through regulating PI3K/Akt signaling\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-08-28 16:12:26\",\"doi\":\"10.21203/rs.3.rs-4839192/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-09-16T09:46:55+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-09-16T09:39:29+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"281360551827784443180369418599481563341\",\"date\":\"2024-09-14T09:44:56+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-09-01T09:42:06+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-08-02T02:01:18+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-08-02T01:15:31+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Journal of Orthopaedic Surgery and Research\",\"date\":\"2024-08-01T04:26:27+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-orthopaedic-surgery-and-research\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"josr\",\"sideBox\":\"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)\",\"snPcode\":\"13018\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13018/3\",\"title\":\"Journal of Orthopaedic Surgery and Research\",\"twitterHandle\":\"@MSKmedBMC\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"6c1ca1d7-4fa7-4266-8065-4f69b97af81f\",\"owner\":[],\"postedDate\":\"August 28th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-01-06T16:00:28+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4839192\",\"link\":\"https://doi.org/10.1186/s13018-024-05270-1\",\"journal\":{\"identity\":\"journal-of-orthopaedic-surgery-and-research\",\"isVorOnly\":false,\"title\":\"Journal of Orthopaedic Surgery and Research\"},\"publishedOn\":\"2024-12-31 15:56:58\",\"publishedOnDateReadable\":\"December 31st, 2024\"},\"versionCreatedAt\":\"2024-08-28 16:12:26\",\"video\":\"\",\"vorDoi\":\"10.1186/s13018-024-05270-1\",\"vorDoiUrl\":\"https://doi.org/10.1186/s13018-024-05270-1\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4839192\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4839192\",\"identity\":\"rs-4839192\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}